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Over the last 2 decades, yeast two-hybrid became an invaluable technique to decipher protein-protein interaction networks. In the field of virology, it has proven instrumental to identify virus-host interactions that are involved in viral embezzlement of cellular functions and inhibition of immune mechanisms. Here, we present a yeast two-hybrid protocol that has been used in our laboratory since 2006 to search for cellular partners of more than 300 viral proteins. Our aim was to develop a robust and straightforward pipeline, which minimizes false-positive interactions with a decent coverage of target cDNA libraries, and only requires a minimum of equipment. We also discuss reasons that motivated our technical choices and compromises that had to be made. This protocol has been used to screen most non-structural proteins of murine hepatitis virus (MHV), a member of betacoronavirus genus, against a mouse brain cDNA library. Typical results were obtained and are presented in this report.

Over the last 2 decades, yeast two-hybrid became an invaluable technique to decipher protein-protein interaction networks. In the field of virology, it has proven instrumental to identify virus-host interactions that are involved in viral embezzlement of cellular functions and inhibition of immune mechanisms. Here, we present a yeast two-hybrid protocol that has been used in our laboratory since 2006 to search for cellular partners of more than 300 viral proteins. Our aim was to develop a robust and straightforward pipeline, which minimizes false-positive interactions with a decent coverage of target cDNA libraries, and only requires a minimum of equipment. We also discuss reasons that motivated our technical choices and compromises that had to be made. This protocol has been used to screen most non-structural proteins of murine hepatitis virus (MHV), a member of betacoronavirus genus, against a mouse brain cDNA library. Typical results were obtained and are presented in this report.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that has been responsible for an epidemic outbreak of unprecedented magnitude in recent years. Since then, significant efforts have been made to better understand the biology of this virus but we still have a poor knowledge of CHIKV interactions with host cell components at the molecular level. Here we describe the extensive use of high-throughput yeast two-hybrid (HT-Y2H) to characterize interactions between CHIKV and human proteins. A total of 22 high-confidence interactions, which essentially involved the viral non-structural protein nsP2, were identified and further validated by protein complementation assay (PCA). These results were integrated to a larger network obtained by extensive mining of literature for alphavirus-host interactions. To investigate the role of cellular proteins interacting with nsP2, gene silencing experiments were performed in cells infected by a recombinant CHIKV expressing Renilla luciferase as a reporter. Collected data involve heterogeneous nuclear ribonucleoprotein K (hnRNP-K) and ubiquilin 4 (UBQLN4) in CHIKV replication in vitro. In addition, we showed that CHIKV nsP2 induces a cellular shut-off as previously reported for other Old-World alphaviruses, and determined that among binding partners identified by yeast two-hybrid, the tetratricopeptide repeat protein 7B (TTC7B) plays a significant role in this activity. Altogether, this report provides a first interaction map between CHIKV and human proteins, and involves new host cell proteins in the replication cycle of this virus., info:eu-repo/semantics/published

Viruses have evolved various strategies to escape the antiviral activity of type I interferons (IFN-alpha/beta). For measles virus, this function is carried by the polycistronic gene P that encodes, by an unusual editing strategy, for the phosphoprotein P and the virulence factor V (MV-V). MV-V prevents STAT1 nuclear translocation by either sequestration or phosphorylation inhibition, thereby blocking IFN-alpha/beta pathway. We show that both the N- and C-terminal domains of MV-V (PNT and VCT) contribute to the inhibition of IFN-alpha/beta signaling. Using the two-hybrid system and co-affinity purification experiments, we identified STAT1 and Jak1 as interactors of MV-V and demonstrate that MV-V can block the direct phosphorylation of STAT1 by Jak1. A deleterious mutation within the PNT domain of MV-V (Y110H) impaired its ability to interact and block STAT1 phosphorylation. Thus, MV-V interacts with at least two components of IFN-alpha/beta receptor complex to block downstream signaling.